Daprodustat prevents cyclosporine-A–mediated anemia and peritubular capillary loss
Authors: Robert Labes, Lennart Brinkmann, Vera A. Kulow, Kameliya Roegner, Susanne Mathia, Björn Balcerek, Pontus B. Persson, Christian Rosenberger, Michael Fähling
Journal: Kidney International (2022)

Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A. Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A. Translational StatementCyclosporine-A (CsA) treatment per se may cause anemia, which is amenable to intervention with the hypoxia-inducible factor activator daprodustat. The latter is currently under US Food and Drug Administration and European Medicines Agency review for treatment of renal anemia. Long-term daprodustat therapy offsets most of the CsA-induced protein phosphorylation changes that are key for immunosuppression, but probably also for adverse effects, like kidney toxicity. Early CsA-induced kidney toxicity presents with diminished kidney capillaries, which are restored by daprodustat. CsA may inhibit angiogenesis (e.g., through dephosphorylation of protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]). In kidney biopsies, reduced phospho-PRKAR2A may represent an early sign of CsA toxicity. Cyclosporine-A (CsA) treatment per se may cause anemia, which is amenable to intervention with the hypoxia-inducible factor activator daprodustat. The latter is currently under US Food and Drug Administration and European Medicines Agency review for treatment of renal anemia. Long-term daprodustat therapy offsets most of the CsA-induced protein phosphorylation changes that are key for immunosuppression, but probably also for adverse effects, like kidney toxicity. Early CsA-induced kidney toxicity presents with diminished kidney capillaries, which are restored by daprodustat. CsA may inhibit angiogenesis (e.g., through dephosphorylation of protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]). In kidney biopsies, reduced phospho-PRKAR2A may represent an early sign of CsA toxicity. The immunosuppressant cyclosporine-A (CsA) acts through inhibition of the phosphatase calcineurin. CsA is standard of care in organ transplantation and in a variety of autoimmune diseases.1Azzi J.R. Sayegh M.H. Mallat S.G. Calcineurin inhibitors: 40 years later, can't live without.J Immunol. 2013; 191: 5785-5791Crossref PubMed Scopus (230) Google Scholar, 2Issa N. Kukla A. Ibrahim H.N. 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Scheidl J. et al.Cyclosporin a induces renal episodic hypoxia.Acta Physiol (Oxf). 2017; 219: 625-639Crossref PubMed Scopus (23) Google Scholar Interestingly, some groups report anemia under CsA treatment,9Bardet V. Junior A.P. Coste J. et al.Impaired erythropoietin production in liver transplant recipients: the role of calcineurin inhibitors.Liver Transpl. 2006; 12: 1649-1654Crossref PubMed Scopus (16) Google Scholar,10Lei D.M. Piao S.G. Jin Y.S. et al.Expression of erythropoietin and its receptor in kidneys from normal and cyclosporine-treated rats.Transplant Proc. 2014; 46: 521-528Crossref PubMed Scopus (6) Google Scholar as well as relatively low erythropoietin (EPO) blood levels.11Sinkeler S.J. Zelle D.M. Homan van der Heide J.J. et al.Endogenous plasma erythropoietin, cardiovascular mortality and all-cause mortality in renal transplant recipients.Am J Transplant. 2012; 12: 485-491Crossref PubMed Scopus (19) Google Scholar Although the mechanisms of CsA-induced anemia are unclear, it can be assumed that anemia contributes to CsA nephrotoxicity, by limiting oxygen delivery. Hence, anemia and kidney insufficiency may create a vicious cycle. Small, highly selective inhibitors of prolyl-hydroxylase domain, like daprodustat (GSK-1278863), emerge as erythropoiesis-stimulating agents, based on their ability to upregulate hypoxia-inducible factor (HIF), and hence, EPO. Herein, we test daprodustat's potential to alleviate both CsA-induced anemia and kidney toxicity. Moreover, in mice treated for up to 8 weeks with CsA, daprodustat, or daprodustat/CsA, phospho-proteomics and subsequent factor analysis assess the fingerprint of early CsA-induced kidney toxicity, as well as potential treatment options. Remarkably, through mechanisms yet unclear, daprodustat virtually offsets CsA-induced changes in protein phosphorylation. Daprodustat prevents CsA-induced kidney capillary rarefaction and anemia. Animal experiments were approved by local authorities (Landesamt für Gesundheit und Soziales: G0227-17) and performed according to the guidelines of the American Physiological Society. Mice (C57BL/6N, male, 10 weeks old) were obtained from Janvier and had free access to chow and water. Seven days before treatment, mice received a low-salt diet (≤0.03% sodium) and distilled water until termination. Daily doses of CsA (80 mg/kg), daprodustat (10 mg/kg), or a combination of both were given by oral gavage for 4 or 8 weeks. Control mice were treated with the vehicle methylcellulose. Blood samples were collected from the submandibular vein.12Golde W.T. Gollobin P. Rodriguez L.L. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.Lab Anim (NY). 2005; 34: 39-43Crossref PubMed Scopus (333) Google Scholar Kidneys were either fixed in 4% paraformaldehyde (24 hours, room temperature), dehydrated, and embedded in paraffin for histologic analysis or snap frozen in liquid nitrogen for molecular analyses. Plasma CsA and plasma creatinine were measured by Labor Berlin–Charité Vivantes GmbH. Hemoglobin (Hb) concentration was measured using an ABL800 Flex blood gas analyzer (Radiometer GmbH). Liquid nitrogen-frozen kidney samples were ground to fine powder. RNA was extracted using RNA-Bee (Biozol Diagnostica Vertrieb GmbH), according to the manufacturer's instructions. RNA quantity and quality were estimated using a NanoDrop 2000 (Thermo Fisher Scientific Inc.). cDNA synthesis was performed using random primers and Superscript II reverse transcriptase (Thermo Fisher Scientific Inc.). Quantitative polymerase chain reaction was performed using the CFX Connect cycler (Bio-Rad Laboratories, Inc.) and SYBR Green Master Mix (Thermo Fisher Scientific Inc.; number 4367659). Primers were designed using the primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index) and obtained from TIB Molbiol Syntheselabor GmbH. All primers used are listed in Supplementary Table S1. The polymerase chain reaction protocol consisted of a 10-minute holding step at 95 °C and 40 cycles of 20 seconds at 95 °C and 40 seconds at 60 °C. Samples were measured in triplicate. The triplicate's arithmetic means were normalized to the housekeeping gene β-actin by employing the ΔCt-method. Liquid nitrogen-frozen kidney samples were ground to fine powder and resolved in lysis buffer (50 mM Tris, pH 6.8, 4 M urea, 1% sodium dodecylsulfate, and 12.5 mM dithiothreitol). Protein concentration was measured with a NanoDrop 2000 (Thermo Fisher Scientific Inc.). Proteins were separated by sodium dodecylsulfate– polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature with 5% skimmed milk in triethanolamine-buffered saline with Tween 20. Membranes were probed with primary antibodies (protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]: number 612243, BD Biosciences; phosphorylated PRKAR2A: number sc-377575, Santa Cruz Biotechnology; HIF-1α: number 10006421, Cayman Chemical) at 4 °C overnight. Primary antibodies were detected using horseradish peroxidase conjugated secondary antibodies (PRKAR2A and phospho-PRKAR2A: horseradish peroxidase–donkey anti-mouse IgG, number 715-035-150; HIF-1α: horseradish peroxidase–donkey anti-rabbit IgG, number 711-035-152; Jackson ImmunoResearch Europe Ltd) for 1 hour at room temperature. Visualization was performed by chemiluminescence solution (WesternBright Chemilumineszenz Substrat; Biozym Scientific GmbH), according to the manufacturer's instruction in a Chemostar Imager (Intas Science Imaging Instruments GmbH). Quantification of protein signals was done with Image Studio Lite software (LI-COR Biosciences). For histologic analyses, paraffin-embedded kidney samples were cut (1.5 μm), deparaffinized with xylene (30 minutes, room temperature), and rehydrated in a decreasing series of ethanol washes ending with double-distilled water and then subjected to either periodic acid–Schiff staining or immunofluorescence staining. Images were recorded with an Eclipse Ti2-A microscope and a DS-Ri2 camera controlled by the NIS-Elements software (Nikon). Large images were recorded as separate images and stitched afterwards. Periodic acid–Schiff staining was performed according to standard procedures (ISBN: 978-3-8274-2254-5). Briefly, deparaffinized and rehydrated slices were incubated in 0.5% periodic acid, rinsed with tap water, and incubated in Schiff reagent. After rinsing with tap water, slices were counterstained with Mayer hematoxylin and washed in tap water. Slices were dehydrated and mounted with a synthetic mounting medium. For immunofluorescence staining, antigens were retrieved with Target Retrieval Solution (Agilent Technologies, Inc.) in a pressure cooker (12 minutes) and unspecific binding sites were blocked with 5% skim milk in triethanolamine-buffered saline with Tween 20 (1 hour, room temperature). Primary and secondary antibodies were diluted in blocking solution and incubated at 4 °C overnight or 1 hour at room temperature. For detection of endothelial cells, a monoclonal rat antibody against CD31 was used (number DIA-310; Dianova GmbH). Monoclonal rabbit antibody against vimentin (number ab92547; Abcam) and polyclonal goat antibodies against neutrophil gelatinase-associated lipocalin (NGAL; number AF1857; R&D Systems) and kidney injury molecule-1 (KIM-1; number AF1817; R&D Systems) were used as kidney damage markers. To assess the amount of vimentin-stained structures and capillary density (CD31) in kidney slices, the area of the immunofluorescence signal was measured. All images were analyzed with the ImageJ distribution Fiji.13Schindelin J. Arganda-Carreras I. Frise E. et al.Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (33784) Google Scholar,14Rueden C.T. Schindelin J. Hiner M.C. et al.ImageJ2: ImageJ for the next generation of scientific image data.BMC Bioinformatics. 2017; 18: 529Crossref PubMed Scopus (3527) Google Scholar Glomeruli were separated by hand. Stitched images were normalized with the filter Normalize Local Contrast, blurred, and thresholded by build-in automatic thresholding procedures. The area of the thresholded image was measured and divided through total kidney area or glomerular area, respectively. For detailed settings, see Supplementary Table S2. Phospho-proteomic analysis was performed using the scioPhospho array (Sciomics GmbH). Proteins were extracted from fresh snap-frozen kidney samples with scioExtract buffer, according to standard operation procedures. Samples were labeled at an adjusted protein concentration with scioDye 2. After 2 hours, excess dye was removed and the buffer was exchanged to phosphate-buffered saline. All labeled samples were stored at –20 °C. Samples were analyzed on scioPhospho antibody microarrays. In brief: 1360 different proteins were targeted with 1830 antibodies, whereas each antibody was represented by 4 replicates. After the arrays were blocked with scioBlock on a Hybstation 4800 (Tecan Group AG), the samples were incubated to scioPhosphomix 1. This procedure made it possible to attain information on overall phosphorylation levels at serine, threonine, and tyrosine residues for all 1360 proteins analyzed. Slides were incubated for 3 hours before being washed with 1 × phosphate-buffered saline with Tween 20 and rinsed with 0.1 × phosphate-buffered saline and water. Subsequently, samples were dried with nitrogen. All slides were scanned with identical laser power and adjusted photomultiplier tube settings using a Powerscanner (Tecan Group AG). Spot segmentation was performed using a GenePix Pro 6.0 (Molecular Devices, LLC). Analysis of the raw data was conducted using the linear models for microarray data package of R-Bioconductor after uploading the median signal intensities. Furthermore, a cyclic Loess (Locally Weighted Scatterplot Smoothing) normalization was applied. Fitting of a 1-factorial linear model with linear models for microarray data resulted in a 2-sided t test or F-test based on moderated statistics. The false discovery rate, according to Benjamini and Hochberg, was employed to adjust the P values for multiple testing. Proteins were deemed differential if the log fold change >0.25 and the adjusted P value was <0.05. Identified differences in protein abundance and phosphorylation levels between samples or sample groups are presented as log fold change calculated for the basis 2. Raw data of scioPhospho proteomic assay are available as online supplement. Gene set enrichment analysis was performed with the SetRank package15Simillion C. Liechti R. Lischer H.E. et al.Avoiding the pitfalls of gene set enrichment analysis with SetRank.BMC Bioinformatics. 2017; 18: 151Crossref PubMed Scopus (60) Google Scholar for R (version 3.6.2; R Core Team, 2019), according to the provided instructions. Annotation tables were used from Gene Ontology.16Ashburner M. Ball C.A. Blake J.A. et al.The Gene Ontology Consortium. Gene ontology: tool for the unification of biology.Nat Genet. 2000; 25: 25-29Crossref PubMed Scopus (28424) Google Scholar,17Gene Ontology ConsortiumThe Gene Ontology resource: enriching a GOld mine.Nucleic Acids Res. 2021; 49: D325-D334Crossref PubMed Scopus (1425) Google Scholar All tested proteins were used as background set. The set collection was created with a maxSetSize of 500, and SetRank analysis was performed using ranks and a false discovery rate cutoff of 0.05. Statistical analysis was performed with GraphPad Prism 8 software. Outliers were identified by the robust regression and outlier removal (ROUT) method (Q = 5%) and removed for further analysis.18Motulsky H.J. Brown R.E. Detecting outliers when fitting data with nonlinear regression - a new method based on robust nonlinear regression and the false discovery rate.BMC Bioinformatics. 2006; 7: 123Crossref PubMed Scopus (863) Google Scholar Normal distribution was tested with the Kolmogorov-Smirnov test. If data were normally distributed and equal SD could be assumed (largest SD difference < 2-fold), ordinary 1-way analysis of variance was used, followed by the Tukey post hoc test. If equal SD could not be assumed (largest SD difference > 2-fold), the Brown-Forsythe analysis of variance was performed, followed by the Dunnett T3 post hoc test. For data that did not follow normal distribution, the nonparametric Kruskal-Wallis test with Dunn post hoc test was used. The Student t test (normal distribution) or Mann-Whitney test (no normal distribution) served for significance analysis of 2 groups. All data were visualized as box plots with median, lower quartile, and upper quartile. Whiskers show the minimum and maximum range of the values. P < 0.05 was considered significant. Whole blood trough levels were 4193 ± 587 ng/ml at 4 weeks and 3512 ± 378 ng/ml at 8 weeks in the CsA group. In the daprodustat/CsA group, CsA trough levels were 3270 ± 579.6 ng/ml at 4 weeks (P = 0.0208 vs. CsA alone) and 3130 ± 335 ng/ml at 8 weeks (not significant vs. CsA alone) (Supplementary Figure S1). CsA decreased blood Hb at 8 weeks (16.13 ± 0.58 vs. 14.72 ± 0.39 g/dl; P = 0.0008). Compared with controls, daprodustat alone showed no significant increase. However, when comparing with CsA treatment, daprodustat/CsA significantly increased Hb: 14.32 ± 1.22 versus 18.28 ± 0.83 g/dl (P = 0.0096) at 4 weeks and 14.72 ± 0.39 versus 17.38 ± 0.46 g/dl (P < 0.0001) at 8 weeks (Figure 1a and b). Hematocrit measurement rendered comparable results (Supplementary Figure S2). CsA-induced anemia likely was not due to lack of HIF and EPO, because HIF target genes were upregulated (Supplementary Figure S3). CsA-induced kidney injury was assessed by plasma creatinine, routine histology, and kidney injury markers. As shown in Figure 1c and d, plasma creatinine remained unchanged: 0.22 ± 0.03 versus 0.28 ± 0.08 mg/dl (P = 0.5536) at 4 weeks and 0.29 ± 0.09 versus 0.28 ± 0.14 mg/dl (P = 0.9951) at 8 weeks. Daprodustat or daprodustat/CsA had no impact on plasma creatinine (4 weeks: daprodustat = 0.23 ± 0.07 mg/dl; daprodustat/CsA = 0.19 ± 0.02 mg/dl; 8 weeks: daprodustat = 0.27 ± 0.14 mg/dl; daprodustat/CsA = 0.40 ± 0.87 mg/dl). However, following CsA, periodic acid–Schiff staining revealed focal tubular atrophy and interstitial fibrosis (Supplementary Figure S4). KIM-1, indicating proximal tubular stress,19Han W.K. Bailly V. Abichandani R. et al.Kidney injury molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury.Kidney Int. 2002; 62: 237-244Abstract Full Text Full Text PDF PubMed Scopus (1415) Google Scholar was upregulated at 4 and 8 weeks of CsA and similarly with daprodustat/CsA (Figure 2a and b). NGAL, indicating distal nephron injury,20Wen Y. Parikh C.R. Current concepts and advances in biomarkers of acute kidney injury.Crit Rev Clin Lab Sci. 2021; 58: 354-368Crossref PubMed Scopus (44) Google Scholar,21Bolignano D. Donato V. Coppolino G. et al.Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage.Am J Kidney Dis. 2008; 52: 595-605Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar seemed upregulated at 4 weeks, with high interindividual variation, but the difference did not reach statistical significance. However, at 8 weeks of CsA, Ngal levels were significantly increased, whereas daprodustat did not changed the CsA effect (Figure 2c and d). Dickkopf-3 (Dkk-3) is a stress-induced tubular epithelia–derived profibrotic glycoprotein and, thus, novel marker of chronic and progressive kidney disease.22Zewinger S. Rauen T. Rudnicki M. et al.Dickkopf-3 (DKK3) in urine identifies patients with short-term risk of eGFR loss.J Am Soc Nephrol. 2018; 29: 2722-2733Crossref PubMed Scopus (60) Google Scholar, 23Fang X. Hu J. Chen Y. et al.Dickkopf-3: current knowledge in kidney diseases.Front Physiol. 2020; 11533344Crossref Scopus (21) Google Scholar, 24Schunk S.J. Speer T. Petrakis I. et al.Dickkopf 3-a novel biomarker of the "kidney injury continuum.".Nephrol Dial Transplant. 2021; 36: 761-767Crossref PubMed Scopus (21) Google Scholar Indeed, at both 4 and 8 weeks of CsA, Dkk-3 was significantly upregulated, whereas daprodustat/CsA showed no alteration compared with control (Figure 2e and f). Vimentin is a typical cytoskeleton filament of mesenchymal cells. During renal scarring, vimentin may occur de novo in the nephron, thus fueling the concept of epithelial-to-mesenchymal transdifferentiation.25Lovisa S. Zeisberg M. Kalluri R. Partial epithelial-to-mesenchymal transition and other new mechanisms of kidney fibrosis.Trends Endocrinol Metab. 2016; 27: 681-695Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar Although this concept could not be proven by lineage tracing,26Kriz W. Kaissling B. Le Hir M. Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy?.J Clin Invest. 2011; 121: 468-474Crossref PubMed Scopus (368) Google Scholar tubular vimentin expression may indicate a profibrotic epithelial phenotype. Immunohistochemistry revealed focal vimentin upregulation in the superficial and mid cortex at 4 weeks of CsA (Figure 3a and b). Signals typically located in both interstitial and tubular cells arranged in stripes toward the medulla. Supporting, kidney homogenates exhibited elevated vimentin mRNA (Figure 3c). In double immunostaining, vimentin colocated with KIM-1, but not NGAL, in tubules (Figure 4). KIM-1 seemed to occur in proximal, and NGAL in distal, tubules, judged by presence or absence of brush border and their general appearance. Interstitial vimentin mostly occurred in the vicinity of KIM-1–positive, but much less in that of NGAL-positive, tubules. These data are consistent with earlier findings of Ichimura et al., showing KIM-1 and vimentin colocalization in damaged tubules of the postischemic kidney.27Ichimura T. Bonventre J.V. Bailly V. et al.Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury.J Biol Chem. 1998; 273: 4135-4142Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar Daprodustat/CsA tended to reduce signal intensity, although signal abundance, the criterion chosen for semiquantitation, remained unchanged. Tubular vimentin expression was transient, and no longer detectable after 8 weeks of CsA at the protein level, albeit Vim mRNA was still significantly elevated (Supplementary Figure S5).Figure 4Colocalization of vimentin (VIM) and kidney injury markers. Mice were treated for 4 weeks with vehicle (controls), cyclosporine-A (CsA), daprodustat (Dapro), or Dapro/CsA. Shown is the coimmunofluorescence for VIM and kidney injury molecule-1 (KIM-1) (a) or neutrophil gelatinase-associated lipocalin (NGAL) (b). Controls and Dapro-treated kidneys expressed neither KIM-1 nor NGAL. In both CsA groups, VIM appeared de novo in KIM-1–positive tubules and was upregulated in the interstitium surrounding KIM-1– or NGAL-positive tubules. Bars = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Taken together, based on molecular injury markers and histology, we generated early CsA-induced kidney injury. Because CsA principally inhibits the phosphatase calcineurin, alterations of protein phosphorylation likely are involved in CsA-induced kidney injury. For in-depth analysis of the phospho-proteome, we chose a commercial assay (scioPhospho; Sciomics GmbH) covering 1360 factors involved in tissue injury and repair, including T-cell activation, cell cycle regulation, oxidative stress response, cell adhesion, apoptosis, and angiogenesis (Figure 5a). The analysis was conducted after 8 weeks of CsA, daprodustat, or their combination, when CsA trough levels were comparable between treatment groups. First, compared with controls, CsA changed expression of 79, the phosphorylation of 86, and both expression and phosphorylation in 13 proteins (Figure 5b). Daprodustat alone had limited effect, with only 2 proteins changed at the phosphorylation level (Figure 5c). However, daprodustat virtually abolished the effect of CsA on protein phosphorylation, as only 6 of previously 86 proteins remained changed (Figure 5d). Second, compared with CsA, CsA/daprodustat changed the expression of 47, the phosphorylation of 30, and both expression and phosphorylation of 2 proteins (Figure 5e). It is important to recognize that in the Venn diagrams, both upregulated and downregulated candidates were included. CsA favored both protein expression and phosphorylation (see volcano plots in Supplementary Figure S6A and B). Daprodustat alone had virtually no effect on protein expression or phosphorylation (Supplementary Figure S6C and D). Compared with CsA, daprodustat/CsA had no gross effect on protein expression, but overall favored dephosphorylation and virtually silenced changes in protein phosphorylation (Supplementary Figure S6E to H). Next, we extracted the top regulated proteins: CsA led to upregulation of profibrotic factors DKK-3, transforming growth factor-β2, and fibroblast growth factor, which is in line with CsA promoting kidney fibrosis (Tables 1 and 2). These factors, however, were not changed by daprodustat. Furthermore, CsA favored dephosphorylation, and hence silencing of key angiogenic factor PRKAR2A,28Bir S.C. Xiong Y. Kevil C.G. et al.Emerging role of PKA/eNOS pathway in therapeutic angiogenesis for ischaemic tissue diseases.Cardiovasc Res. 2012; 95: 7-18Crossref PubMed Scopus (81) Google Scholar whereas daprodustat/CsA reversed this effect. Daprodustat/CsA also reduced expression of CsA binding protein cyclophilin (peptidylprolyl isomerase A).29Willenbrink W. Halaschek J. Schuffenhauer S. et al.Cyclophilin A, the major intracellular receptor for the immunosuppressant cyclosporin A, maps to chromosome 7p11.2-p13: four pseudogenes map to chromosomes 3, 10, 14, and 18.Genomics. 1995; 28: 101-104Crossref PubMed Scopus (19) Google ScholarTable 1Top regulated kidney proteins in chronic CsA toxicitySymbolProtein nameLogFCAdjusted P valueFunction/remarksTop regulated candidates: CsA vs. control: expression CA9 (CAR9)Carbonic anhydrase 91.260.0156HIF1-α target gene, acid-base balance ULBP1UL16-binding protein 11.03<0.0001Natural killer cell cytotoxicity CD55Complement decay-accelerating factor1.01<0.0001Inhibition of complement pathway TNFRSF11BTNF receptor superfamily member 11B0.99<0.0001Tumor necrosis factor signaling DKK3Dickkopf-related protein 30.97<0.0001Kidney damage marker IL19Interleukin-190.910.0495Cytokine,